Functional analysis of the promoter and first intron of the human lysyl oxidase gene

Abstract

Alterations in the synthesis and activity of lysyl oxidase occur concomitant with developmental changes in collagen and elastin deposition and with the pathogenesis of several acquired and heritable connective tissue disorders. To begin to unravel the mechanisms that control lysyloxidase gene expression, we have previously reported the complete exon-intron structure of the human lysyl oxidase gene. We have now sequenced this entire gene, including all six introns and 4 kb of DNA 5′ of exon 1. Analysis of over 13 kb of intervening sequence and 5′ flanking sequence revealed a concentration of conserved consensus sequence elements within the first intron and 1 kb immediately 5′ of exon 1. Analysis of intron 1 and the 5′ flanking domain, using recombinant plasmids containing the chloramphenicol acetyl transferase (CAT) reporter gene, identified functional DNA sequence elements within these non-coding domains responsible for inhibition and up-regulation of CAT activity in primary cultures of human skin fibroblasts, in smooth muscle cells, revertant cells derived from an osteosarcoma cell line and malignant c-Ha-ras-transformed osteosarcoma cells. DNA sequence elements within intron 1, in particular, resulted in a marked increase in CAT reporter activity in cultured fibroblasts, smooth muscle cells and osteosarcoma cells. In c-Ha-ras-transformed osteosarcoma cells, however, no such enhancer activity of intron 1 sequence was observed. Ras-transformed osteosarcoma cells exhibited reduced steady-state levels of lysyl oxidase mRNA that was primarily controlled through reduced transcription of the lysyl oxidase gene. The lack of any up-regulation of CAT activity in these ras-transformed cells by sequence elements within intron 1 suggests a complex interaction between cis-acting domains and trans-acting transcriptional factors in the 5′ promoter domain and the first intron of the lysyl oxidase gene.